Abstract Chromatin is the natural substrate for DNA transactions in eukaryotic cells. Studies from the past two decades have shown that chromatin can be modified through ATP-dependent chromatin remodeling and histone modification. Although the role of chromatin remodeling in transcription has been well established, its involvement in other DNA transactions, such as DNA repair, checkpoint regulation and replication is relatively unclear. We and others have shown that the evolutionarily conserved INO80 chromatin remodeling complex is directly involved in double strand break (DSB) repair, checkpoint regulation, as well as DNA replication. Despite the initial characterization, the precise mechanisms by which INO80 participates in these distinct processes remain unclear. INO80 can be recruited to a conventional DSB through its interaction with phosphorylated H2A (3-H2AX). The Nhp10 subunit of INO80 is required for this interaction, suggesting that INO80 can employ a specific subunit to target its function to conventional DSB repair. Recently, we have also shown that INO80 is involved in checkpoint regulation through Mec1/Tel1 phosphorylation of its Ies4 subunit. However, the specific functions of Ies4 phosphorylation in DNA replication and intra-S phase checkpoint remain to be elucidated. Moreover, INO80 has also been implicated in DNA replication recently, and our preliminary studies suggest that INO80 plays a specific role in DNA damage tolerance during replication. The distinct involvement of a single complex in multiple DNA transactions suggests that INO80 utilizes sophisticated regulatory mechanisms in genome maintenance. We hypothesize that INO80 is a key regulator of genome maintenance and employs distinct mechanisms to target and participate in DNA repair, checkpoint regulation and DNA replication. The main goal of our proposal is to test this hypothesis by dissecting the mechanisms of INO80 in distinct processes involved in genome maintenance. We will reveal the mechanism of Ies4 phosphorylation in replication and checkpoint regulation, determine the mechanism targeting INO80 to replication and elucidate the mechanism of INO80 function in DNA damage tolerance. These studies will provide significant new insights into how chromatin remodeling is involved in fundamental biological processes such as DNA repair and replication. Given that defects in DNA replication and repair can cause genome instability and cancer, our study may reveal new mechanisms for tumorigenesis and provide potential novel targets for cancer detection and treatment.